2.1. CHIMERIC CYTOKINES
Certain cells of the body are capable of producing factors, called cytokines, which act as messengers and communicate with other cells, thereby coordinating biological functions. For example, lymphocytes may produce lymphokines, factors which interact with various components of the immune system in order to effectively orchestrate the immune response. Neurotrophic factors are cytokines which can promote the survival and/or differentiation of components of the nervous system.
As intercellular messengers, cytokines typically interact with specific populations of cells via cytokine receptor molecules. Accordingly, a cytokine is targeted toward particular receptor-bearing cells. It has been shown that cytokines can be used to deliver toxic substances to a cell population by linking the cytokine to the toxic substance. For example, Siegall et al. (1989, Fed. Am. Soc. Exp. Biol. 3:2647-2652) fused a cDNA encoding the cytokine transforming growth factor alpha to the 5' end of a gene encoding a modified form of Pseudomonas exotoxin A which was devoid of the cell recognition domain. The resulting chimeric molecule was expressed in Escherichia coli, isolated, and found to be extremely cytotoxic to cells specifically displaying the epidermal growth factor receptor. Ogata et al. (1989, Proc. Natl. Acad. Sci. U.S.A. 86:4215-4219) produced a recombinant chimeric toxin in which the binding cell domain of Pseudomonas exotoxin (PE) was replaced by the murine lymphokine interleukin 4 (IL-4); the chimeric protein, IL-4-PE40, was found to be cytotoxic to murine IL-4 receptor-bearing cell lines. Banker et al. (1989, J. Cell Physiol. 139:51-57) describe an epidermal growth factor-ricin A chain chimera. Williams et al. (1987, Protein Eng. 1:493-498) replaced the diphtheria toxin receptor binding domain with a synthetic gene encoding interleukin 2 (IL-2) and a translational stop signal. The diphtheria toxin/IL-2 fusion protein was found to selectively inhibit protein synthesis in IL-2 receptor bearing cells, whereas cell lines which did not express the IL-2 receptor were resistant to toxin action.
Other investigators have constructed recombinant DNA molecules which comprise a cytokine gene as well as at least a portion of a bacterial gene. Dicou et al. (1989, J. Neurosci. Res. 22:13-19) fused the complete mouse prepro-nerve growth factor DNA to the carboxyl terminus of the beta-galactosidase gene of Escherichia coli, and also fused a genomic DNA fragment corresponding to codons 11 to 106 of the human nerve growth factor gene to the fifth codon of the amino terminus of beta-galactosidase. Both bacterial vectors were associated with the expression of large amounts of the chimeric proteins. Although after bacterial cell lysis most of the chimeric mouse prepro-nerve growth factor appeared to be insoluble, the majority of human chimeric beta-nerve growth factor seemed to exist in the supernatant. Neurotrophic activity was not repoted.
In recent studies, Ibanez et al. (1990, EMBO J. 9:1477-1483) describe studies of nerve growth factor altered by site-directed mutagenesis. Xie et al. (1990, Proc. Nat. Acad. Sci. U.S.A. 87:3180-3184) describe the use of chimeric opiod peptides in the study of opiate receptors. Ray et al. (1990, Mol. Endocrinol. 102:101) discuss alteration in receptor binding specificity of growth hormone specificity resulting from genomic exon exchange. Cunningham et al. (1990, Science 247:1330) systematically substituted portions of the growth hormone in order to identify mutant hormones which would be unable to bind to the growth hormone receptor. Cunningham et al. (1990, Science 247:1461) relates to site directed mutagenesis of prolactin observed to produce a prolactin variant capable of binding to the growth hormone receptor.